AQUATIC BIOLOGY Aquat Biol
Vol. 6: 91–98, 2009 doi: 10.3354/ab00172
Printed August 2009 Published online July 15, 2009
OPEN ACCESS
Bioaccumulation of inorganic Hg by the juvenile cuttlefish Sepia officinalis exposed to 203Hg radiolabelled seawater and food T. Lacoue-Labarthe1, 2, M. Warnau1, 2, F. Oberhänsli2, J.-L. Teyssié2, P. Bustamante1,* 1
Littoral Environnement et Sociétés (LIENSs), UMR 6250, CNRS-Université de La Rochelle, 2 rue Olympe de Gouges, 17042 La Rochelle Cedex 01, France 2 International Atomic Energy Agency, Marine Environment Laboratories, 4 Quai Antoine Ier, 98000 Principality of Monaco
ABSTRACT: Uptake and depuration kinetics of inorganic mercury (Hg) were investigated in the juvenile common cuttlefish Sepia officinalis following exposures via seawater and food using a sensitive radiotracer technique (203Hg). Cuttlefish readily concentrated 203Hg when exposed via seawater, with whole body concentration factors > 260 after only 10 d of exposure. The total Hg accumulated from seawater was depurated relatively fast with a radiotracer biological half-life (Tb !) of 17 d. During both exposure and depuration periods, accumulated Hg was mainly (> 70%) associated with the muscular parts of the cuttlefish. However, the proportion of the whole-body Hg content associated with the digestive gland increased during exposure and depuration phases, suggesting that the metal was transferred from the muscles towards this organ for detoxification. When fed with radiolabelled food, cuttlefish displayed high assimilation efficiency (> 90%) and the metal was found to be mainly located in the digestive gland (60% of the whole Hg content). Nevertheless, high depuration rates resulted in short Tb ! (i.e. 4 d), suggesting that this organ has a major role in Hg detoxification and depuration. Whatever the exposure pathway, a low proportion of Hg (< 2%) was found in the cuttlebone. Assessment of the relative contribution of the dietary and dissolved exposure pathways to inorganic Hg bioaccumulation in juvenile cuttlefish revealed that Hg was mainly accumulated from food, which contributed 77 ± 16% of the global metal bioaccumulation. KEY WORDS: Mercury · Bioaccumulation · Kinetics · Body distribution · Cephalopod · Relative contribution Resale or republication not permitted without written consent of the publisher
INTRODUCTION Mercury (Hg) is one of the metals of highest concern in the marine environment as it is readily methylated by microorganisms, bioaccumulates in marine biota and consistently biomagnifies along the food chain (Cossa et al. 1990). Among marine organisms, most of the available information on Hg is related to fish, mainly because of their importance as a food source for humans. In fish, most of the Hg (> 95%) is methylated and is therefore bioavailable to the upper trophic levels (Bloom 1992). Hence, fish consumption is an important source of Hg for humans (Svensson et al. 1992) and is a particular health concern (Clarkson 1990).
In contrast to fish, information on Hg in cephalopod tissues is scarce, despite the fact that these molluscs represent an increasing component of the world’s fisheries (Boyle & Rodhouse 2006, FAO 2007). In addition, information on Hg in cephalopods is essentially limited to the main species targeted by fisheries and studies mainly report metal levels in edible tissues, i.e. mantle muscle, arms and fins (e.g. Buzina et al. 1989, Sapunar et al. 1989, Plessi et al. 2001). Recently, a study on a large range of cephalopod species from North Eastern Atlantic waters reported that Hg concentrations varied by 2 orders of magnitude among cephalopod species and that the metal was mainly stored in organic form in the muscular tissues (Bustamante et al. 2006). Several
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other studies on cephalopods from the Mediterranean suggest that these molluscs are able to accumulate high Hg concentrations in their tissues (Renzoni et al. 1973, Rossi et al. 1993, Storelli & Marcotrigiano 1999). Various factors are likely to influence Hg concentrations in cephalopods among which the animal size seems to be of primary importance (Monteiro et al. 1992, Rossi et al. 1993, Pierce et al. 2008). Although it has been suggested that food is the main source of Hg accumulation in cephalopod tissues (Bustamante et al. 2006), the relative contribution of dietary and waterborne pathways has not been assessed in cephalopods. Moreover, as cephalopods are shortlived species, they might be interesting as short-term indicator species for the variation in environmental Hg concentrations (Seixas et al. 2005, Pierce et al. 2008). For these reasons, the aim of the present study was to investigate the biokinetics of Hg uptake and depuration in cephalopods in order to better characterize its bioaccumulation, tissue distribution and retention capacity. The common cuttlefish Sepia officinalis was selected as a model species to study Hg transfer in cephalopods from seawater and food.
MATERIALS AND METHODS Study organisms and radiotracer. Adult cuttlefish were collected by net fishing off Monaco in March and April 2006. They were acclimated and maintained in open-circuit tanks at the International Atomic Energy Agency, Marine Environment Laboratories (IAEAMEL). After mating, the eggs laid by a single female were separated to optimise their oxygenation and kept in aquaria during the whole embryonic development (constantly aerated open circuit; flux: 50 l h–1; salinity: 37 psu; temperature: 17 ± 0.5°C; pH: 8.0 ± 0.1; 12 h light:12 h dark cycle). Hatching occurred approximately 50 d after spawning. Young cuttlefish were then maintained in the same aquarium and fed with brine shrimp Artemia sp. for 5 d before the experiments. The radiotracer 203Hg (as 203HgNO3; half life, T ! = 46.59 d) was purchased from Isotope Products Laboratories. Stock solutions were prepared in 1 N nitric acid to obtain final radioactivity allowing the use of spikes of only a few microliters (typically 5 µl). Experimental procedure. Contamination via seawater: Juveniles (n = 23; mean weight ± SD = 0.258 ± 0.009 g) were placed for 10 d in a 20 l glass aquarium containing 0.45 µm filtered natural seawater (constantly aerated closed circuit; temperature: 17°C; salinity: 37 psu; 12 h light:12 h dark cycle) spiked with 203Hg (0.6 kBq l–1). In terms of stable metal, this concentration corresponded to 20 ng l–1. To facilitate the recur-
rent counting of each individual during the experiment, the juveniles were held individually in separate circular plastic containers (10 × 5 cm, diameter × height) covered with a mesh plastic net to allow for free water circulation. Radiotracer and seawater were renewed every second day to maintain water quality and keep radiotracer activity constant. Radiotracer activities in seawater were checked before and after each water renewal in order to determine the timeintegrated radiotracer activities (Warnau et al. 1996, Rodriguez y Baena et al. 2006a). Juveniles were separated in 2 groups: the first group contained 16 tagidentified individuals and the second was composed of unidentified animals for the body distribution analyses. At different time intervals, radiotracer activities were counted in the same tag-identified juveniles (n = 16) throughout the experiment. According to their physiological state, individuals were removed from the experiment according to the following sampling plan: n = 16 from Days 0 to 3 and n = 14, 7 and 6 at Days 6, 9 and 10, respectively. In addition, after 3 and 9 d of exposure, 3 juveniles of the second group were counted and dissected to determine the radiotracer distribution among the digestive gland, the cuttlebone and the remaining tissues. After this exposure period, the 6 remaining identified and radiolabelled juveniles (n = 6 from Days 0 to 4, n = 4 at Day 6 and n = 1 at Day 11) were held for 11 d in clean, flowing water (open circuit with constant aeration; seawater flux: 50 l h–1; temperature: 17°C; salinity: 37 psu; 12 h light:12 h dark cycle). At different time intervals during the depuration period, the same identified juveniles were counted to establish the depuration kinetics of the radiotracer. Contamination via food: Brine shrimp Artemia sp. were exposed for 5 d in a plastic aquarium containing 4 l of natural seawater spiked with 203Hg until pools of 25 brine shrimp reached 3.1 kBq g–1 wet weight. The organisms were subsequently used as food for the juvenile cuttlefish. As detailed in the seawater experimental procedure, juveniles were separated into 2 groups of identified and non-identified animals which were devoted to the depuration kinetics and body distribution studies, respectively. Hence, 16 newly hatched cuttlefish (mean weight ± SD = 0.297 ± 0.011 g) were placed in individual plastic containers (10 × 5 cm, diameter × height), and held in a 20 l aquarium under the same conditions as in the previous experiment. Each juvenile was fed for 1 h with 25 of the previously radiolabelled Artemia sp. At the end of the feeding period, the cuttlefish were immediately counted. From that time on, the cuttlefish were fed twice a day with uncontaminated Artemia sp. for 1 mo and regularly counted to determine radiotracer depuration kinetics and assimilation efficiency.
Lacoue-Labarthe et al.: Bioaccumulation of inorganic Hg in Sepia officinalis
As mentioned above, juveniles showing poor health condition were removed leading to a sample number decrease throughout the experiment: n = 16 from Days 0 to 2, n = 14 from Days 3 to 14, n = 4 from Days 17 to 22 and n = 1 from Day 24 on. Throughout the depuration period, faeces were removed twice a day to reduce possible radiotracer recycling through leaching from the faeces. In addition, after 3 h, 9 d and 22 d of exposure, 3 other juveniles were counted and dissected to determine the radiotracer distribution among the digestive gland, the cuttlebone and the remaining tissues. Radioanalyses and data treatment. Radioactivities were measured using a high-resolution γ-spectrometry system consisting of 4 coaxial Germanium (N- or Ptype) detectors (EGNC 33-195-R, Canberra® and Eurysis®) connected to a multi-channel analyzer and a computer equipped with spectra analysis software (Interwinner® 6). The detectors were calibrated with an appropriate standard for each counting geometry used and measurements were corrected for background and physical decay of the radiotracer. Counting times were adapted to obtain relative propagated errors less than 5% (Rodriguez y Baena et al. 2006b). They ranged from 10 to 30 min for whole juveniles and from 10 min to 24 h for the dissected tissues. Uptake of 203Hg from seawater was expressed as change in concentration factors (CF; ratio between radiotracer content in the juvenile, Bq g–1, and timeintegrated activity in seawater, Bq g–1) over time (Warnau et al. 1996). Uptake kinetic was best described by a saturation equation: CFt = CFss(1 – e– ket )
(1)
where CFt and CFss are the concentration factors at time t (d) and at steady state (ss), respectively, and k e is the biological depuration rate constant (d–1) (Whicker & Schultz 1982). Radiotracer depuration kinetics were expressed in terms of change in percentage of the remaining activity (i.e. radioactivity at time t divided by initial radioactivity measured in the organisms or in the tissue at the beginning of the depuration period × 100) over time. The depuration kinetic was best fitted by a monoexponential equation: At = A0e– ket
(2)
where At and A0 are the remaining activities (%) at times t (d) and 0, respectively. The determination of ke allows the calculation of the radiotracer biological halflife (Tb ! = ln2/ke). In the context of the seawater and feeding experiments, A0 represents the absorption (A0,w) and the assimilation efficiencies (AE), respectively. Bioaccumulation model. The relative contribution of each uptake pathway was determined using the bio-
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accumulation model originally proposed by Thomann (1981) and revised by Thomann et al. (1995) and Metian et al. (2008). In this model, the total concentration of radiotracers in the juveniles, C t (Bq g–1) is equal to the sum of each concentration resulting from the uptake by the different pathways (Eq. 3): Ct = Cf,ss + Cw,ss
(3)
where Cf,ss is the food-derived radiotracer concentration (Bq g–1) in juveniles at steady state (Eq. 4) and Cw,ss the water-derived radiotracer concentration (Bq g–1) in juveniles at steady state (Eq. 5): Cf,ss = (AE × IR × Cf)/ke,f
(4)
Cw,ss = (A0,w × ku,w × Cw)/ke,w
(5)
where A0,w is the absorption efficiency (%) of the radiotracer from seawater, AE is the assimilation efficiency (%) of the radiotracer from food, Cf and Cw are the radiotracer activities in food and seawater (Bq g–1 and Bq ml–1, respectively), respectively, IR is the ingestion rate (g g–1 d–1), ku,w is the uptake rate constant (d–1) from seawater and ke,f and ke,w are the biological depuration rate constants (d–1) for food and water pathways, respectively. The relative contribution (%) of each uptake pathway is then assessed from the following relationships: % food = C f,ss /(C f,ss + Cw,ss)
(6)
% seawater = Cw,ss/(Cf,ss + Cw,ss)
(7)
Constants (and their statistics) of the best fitting equations (decision based on ANOVA tables for 2 fitted model objects) were estimated by iterative adjustment of the models using the nls curve-fitting routine in R freeware. The level of significance for statistical analysis was set at α = 0.05.
RESULTS Contamination through seawater Uptake activity of 203Hg in whole-body Sepia officinalis was best fitted by a saturation exponential equation with a calculated CFss of 480 (Fig. 1, Table 1). The CF actually measured (mean ± SD) at the end of the uptake period (CF10d) of 203Hg was 260 ± 70 (Table 2). Calculated CF10d for the different organs indicated that 203 Hg was concentrated according to the following decreasing order: digestive gland (1460 ± 480) > remaining tissues (290 ± 80) > cuttlebone (47 ± 9). In terms of body distribution, 203Hg was mainly found in the remaining tissues throughout the exposure period; this compartment accounted for 89 ± 3 and 80 ± 4% of the whole-body load of 203Hg after 3 and 10 d of expo-
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This result indicated that 95% of the 203Hg previously accumulated was depurated with a relatively short biological half-life (i.e. 17 d, Table 1). After 11 d of depuration conditions, most of the 203Hg body load was associated with the remaining tissues (72%) while the digestive gland proportion increased up to 28% (Table 2).
Contamination through food The depuration kinetic of 203Hg ingested with food in Sepia officinalis was best fitted by a mono-exponential model, characterized by a biological half-life of 4 d (Fig. 2, Table 1) and allowed an estimated AE of 91%. During the depuration period, the digestive gland displayed the highest proportion of the total body burden of 203Hg, i.e. 68, 60 and 64% after 3 h, 9 d and 22 d of depuration, respectively (Table 2). The distribution of 203Hg remained unchanged throughout the loss experiment, with the cuttlebone always showing the lowest proportion of the radiotracer (Table 2).
Fig. 1. Sepia officinalis. Whole-body uptake kinetics of 203Hg in juvenile cuttlefish exposed for 10 d to the radiotracer dissolved in seawater. CF: concentration factor; n = 16 from Days 0 to 3 and n = 14, 7 and 6 at Days 6, 9 and 10, respectively
sure, respectively (Table 2). The cuttlebone presented very low Hg activity (
